Since the early 90s more than 400,000 cases of malignant neoplasms are diagnosed in Russia each year. At the same time in Europe, the annual death rate from cancer in the period from 1985 to 2002 continues to grow. Among the causes of mortality cancer occupies second place after diseases of the cardio-vascular system. Therefore the search for new anticancer drugs is one of the most urgent problems of modern biology and medicine.
Currently there is a large number of works devoted to the development of novel drug anticancer drugs based on advances in molecular biology (Richard J P et al., 2003; Takeshima K. et al., 2003; Jyotika A. et al., 2005 Kopnin B P, 2000). It is generally accepted that the fundamental feature of the neoplastic cells is a violation of the regulation of the cell cycle and apoptosis (Chappuis P O, Kapusta L., 2005). It is known that the regulation of cell proliferation processes is controlled by sequential activation of the respective cyclins and cyclin-dependent kinases (CDK).
Cyclin kinase activity is determined by the level of expression of cyclins and relevant activity of specific inhibitors of cyclin kinases (Kastan M V, Bartek J., 2004). There are several families of cyclin kinase inhibitors. The most studied and practically important among them are p16INK4a, p21/CIP/KIP, p27 KIP1 (Lowe S W et al., 2004). Mutations or hypermethylation of promoters of cyclin kinases inhibitor genes are observed in 40-60% of cases of malignant lymphomas, pancreatic cancer and in other malignancies (Sawyers C., 2004; Ortega S. et al, 2002).
Based on these results there was synthesized a number of small molecule inhibitors of cyclin kinases, part of which is now under experimental study (Ross M F, Murphy M R, 2004) and one of which, UCN-1, is in phase one of clinical trials. Another possible direction to generate cyclin kinase inhibitors may be to use functional sequences of the respective intracellular inhibitors (Ziegler A. et al., 2005). Protein p16INK4a is one of the most interesting candidate inhibitors of Cdk (Xu D. et al., 2004; Zhang Y. et al., 2005).
It is known that protein p16INK4a inhibits cyclin D-dependent kinases and thus inhibits G1 phase of the cell cycle (Fu G H et al., 2005; Ben-Saadon R. et al., 2004). It is shown that its function is impaired in a wide range of cancer types (Li J Q et al., 2004). Recently, experimental works started to appear describing the use of gene p16INK4a for gene therapy of tumors of various origins (Lee A W C, Li J H et. Al. 2003; Liu S. X, Tang S. Q, Liang C. Y. 2003; Zhang Y., Liu J. et al. 2005). An additional incentive to seek technologies of applying natural protein proliferation inhibitors was the discovery of short sequences of amino acids (n=15-30) capable of performing vector (transport) functions in respect of peptide sequences and compounds of other chemical nature (RNA, DNA) (Fawell S., Seery J. et al., 1994; Vives E., Brodin P., Lebleu B. 1997; Kaplan I M et al., 2005; Gupta B. et al, 2005; Feraandez-Cameado J. et al., 2005). So far, attempts were made to solve the problem of restoring the impaired function of intracellular proteins by the methods of gene transfer (gene therapy). However, this technology has yet to gain wide use in clinical practice due to a number of fundamental problems.
An alternative way to solve this problem, which is based on the technology of peptide vectors that are able to penetrate cells without damaging the plasma membrane, is very promising due to the weak immunogenicity of such compounds, and the ability to carry quite large molecules.
Combination of capabilities of targeted delivery of peptides into the cell and discovery of short functional domains in protein regulators of various cellular functions set the stage for the design of molecules with pathogenetic orientation (Schutze-Redelmeier M R et al, 2004; Trehin R., Merkle N R, 2004; Cong-Mei Wu et al, 2004). The relative ease of synthesis of these molecules suggests the general possibility for creating individual chemotherapy based on them, i.e., influencing the pathological changes which are characteristic of a particular tumor (Perea S. E. et al, 2004).
The discovery of peptides that are capable to penetrate into the cell without the participation of the membrane proteins and provide for intracellular transport of protein fragments and oligonucleotides linked thereto, opens a new stage in the development of biology and medicine. One of the most effective transporters of large molecules into the cells is the peptide pAntp. Its properties are known, in particular from publications by Derossi D. et al. The third helix of the Antennapedia homeodamain translocates through membranes.//J.Biol. Chem. 269 (1994) 10444-10450 and Morris M C. et al. A peptides carrier for the delivery of biologically active proteins in mammalian cells.//Nat. Biotechnology. 19 (2001) 1173-1176.
The document U.S. Pat. No. 6,569,833 B1 (Cyclacel Limited, GB) discloses peptides that bind to cyclin kinases and include amino acid residues 84-103 of full-chain p16 protein and can be combined with a sequence of a transport protein penetratine by a disulfide bond formed between cysteine residues, especially attached to the C-terminus of the peptide p16 and N-terminus of the peptide pAntp. The disadvantage of this approach is the need for a selective and multi-step synthesis of a chimeric molecule that complicates the way of obtaining the desired product and increases the overall time required for synthesis.
Cited publications indicate that the inhibition of cyclin kinases can be of decisive importance for gene therapy of tumors of various origins. Regarding therapeutic agents based on peptides that bind to cyclin kinase and are combined with a sequence of transport protein, there are certain problems associated with the bioavailability and stability. Therefore, there exists the need to develop new therapeutic agents that have antiproliferative activity against certain types of cancer.
An objective of present invention is to obtain a pharmaceutical composition possessing anti-proliferative and cytotoxic activity due to expressed synergistic effect of a combined use of a chimeric peptide and existing anti-tumor chemotherapeutic agents, such as taxol, 5-fluorouracil, etoposide.
The utility effect of the present invention is an improvement of the biological effect of an agent as compared to solutions known in the art, reflected in the enhancement of cytotoxic and cytostatic effects of the agent on tumor cells.
The utility effect is achieved by a pharmaceutical composition having anti-proliferative and cytotoxic activity, which comprises two active agents, wherein the first active agent is a chimeric peptide comprising a functional sequence of a protein inhibitor of cyclin kinases p16INK4a (SEQ ID NO: 1) of 20 amino acid in length, or an amino acid sequence of protein inhibitor p21/CIP/KIP (SEQ ID NO: 6) and a transport sequence. The second active agent is a chemotherapeutic anticancer agent selected from the group consisting of taxol, 5-fluorouracil, etoposide.
In a preferred embodiment, the pharmaceutical composition is intended for the treatment of cancer selected from the group consisting of colorectal cancer, renal cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer.
In a most preferred embodiment, the pharmaceutical composition may be used for the treatment of colorectal cancer.
In another embodiment, the present invention relates to the use of said pharmaceutical composition for the manufacture of a medicament for the treatment of cancer.
In a preferred embodiment, said pharmaceutical composition can be used for the treatment of cancer selected from the group consisting of colorectal cancer, renal cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer.
In another embodiment, the present invention relates to a method of treating cancer which comprises administering the aforementioned pharmaceutical composition to a mammal in need of such treatment.
In a preferred embodiment of the aforementioned method, the cancer is selected from the group consisting of colorectal cancer, renal cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer.
The following amino acid sequence listings arc part of the disclosure of the present invention:
SEQ ID NO: 1DAAREGFLDTLVVLHRAGAR SEQ ID NO: 2RQIKIWFQNRRMKWKK SEQ ID NO: 3DAAREGFLDTLVVLHRAGARSRQIKIWFQNRRMKWKK SEQ ID NO: 4RGSDAAREGFLDTLVVLHRAGARRQIKIWFQNRRMKWKKSERKRGRQTYT RYQTLELEKEFHFNRYLTRRRRIEIAHALCLTE SEQ ID NO: 5YGRKKRRQRRRG SEQ ID NO: 6PVKRRLDL SEQ ID NO: 7DAAREGFLDTLVVLHRAGARS YGRKKRRQRRRG SEQ ID NO: 8YGRKKRRQRRRGPVKRRLDL
The above enumerated sequence listings are also recorded on a compact disc, which contains a file named “Sequence_Listing.txt” created on Oct. 23, 2013 having a size of 5 KB.